Evolution of the casein multigene family: conserved sequences in the 5'' flanking and exon regions.

The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

The structure of two fast-white myosin heavy chain promoters. A comparative study

Two complete myosin heavy chain genes were isolated from chicken genomic libraries, and shown to code for fast-white isoforms. Isoform specific probes were developed from the 5' nontranslated regions of the two genes and used to identify the developmental stages at which each of the genes are expressed. One of the genes is transcribed in the embryo and the other only in the adult. The 5' flanking regions of the two genes were sequenced along with the first three exons. The 5' untranslated sequences in both genes are not contiguous, one intron is present in the adult gene while the embryonic gene contains two. The promoters of both genes contain the conserved CAAT and TATA box elements observed in other eucaryotic genes. A computer assisted comparison was performed on the two genes at the nucleotide and amino acid levels. No homology could be detected in the 5' flanking regions of the genes except in and around the CAAT and TATA elements, however, structural sequences at the 5' ends were highly conserved as well as the position of the first three introns. The amino acids in and around the ATP binding site are completely conserved between the two isoforms.     

Partial characterization of bovine elastin gene; comparison with the gene for human elastin

A 14 kilobase (kb) genomic clone of the gene for bovine elastin, containing exons 1 and 2, has been characterized. This clone extends about 6.5 kb in the 5' direction from the initiation codon and 978 nucleotides in the 3' direction from exon 2. The size of the first intron is about 6.4 kb. The sequence immediately 5' to the initiation codon is highly conserved between the genes for bovine and human elastins and contains a TATA box consensus sequence (ATAAA), CAAT, and Sp1 binding sites. Several putative AP-2 binding sites are also present. Comparative analysis of the sequences flanking the first exon in the genes for bovine and human elastins identified conserved sequences that may be regulatory control elements. A putative enhancer core sequence is present in the first intron of the genes for bovine and human elastins.     

Structural organization of the bovine thyroglobulin gene and of its 5'-flanking region

The structural organization of the bovine thyroglobulin gene has been investigated by a combination of Southern genomic blotting and direct analysis of cloned gene fragments isolated from a chromosomal DNA library. The entire locus is spread over more than 200,000 base pairs which makes it one of the largest eukaryotic genes studies to date. The coding information is scattered into at least 42 exons, 34 of which have been precisely identified. A different evolutionary origin of the 5' and 3' regions of the gene is supported by the highly different proportion of exonic material they contain (12% and 3%, respectively) and by the existence of sequence homology between the 3' region of thyroglobulin and acetylcholinesterase. Detailed sequence analysis of the 5' region of the gene and its flanking segment demonstrated that a significant homology exists between bovine and human thyroglobulin sequences, except for the presence within the ruminant promoter region of a 220-base-pair sequence belonging to the bovine monomer repeated family.     

Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR

The structure of the 3' one-third of the dystrophin gene has not previously been established. We have used vectorette PCR on a yeast artificial chromosome containing part of the human dystrophin gene to determine that there are 20 exons in this region and to characterize adjacent intron sequences of each one. Combined with previous information on the remainder of the gene, this study shows that the coding sequence is distributed between 79 exons. We have used PCR between exons to measure the distances that separate the more closely clustered exons. Vectorette PCR products were used as probes on Southern blots to assign all the 3' exons to genomic HindIII fragments that are commonly detected in the analysis of dystrophin gene deletions. The results will be useful for determining the effect of genomic deletions on the translational reading frame, for setting up genomic PCR assays to confirm point mutations, for analyzing splice site mutations, and for investigating potential cis-acting elements involved in tissue-specific alternative splicing. Vectorette PCR using primers derived from cDNA sequence represents an efficient and widely applicable method for establishing gene structure and obtaining intron sequence flanking exons, starting from a genomic clone and a cDNA sequence.     

Nucleotide sequence of the gene for human factor IX (antihemophilic factor B)

Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.     

The rat casein multigene family. Fine structure and evolution of the beta-casein gene

Eight overlapping phage clones, spanning 34.4 kilobase pairs of genomic DNA, containing the 7.2-kilobase pair rat beta-casein gene have been isolated and characterized. The first 510 base pairs (bp) of 5' flanking, 110 bp of 3' flanking, and all the exon/intron junctions have been sequenced. The beta-casein gene contains 9 exons ranging in size from 21 to 525 bp. We have attempted to identify potential regulatory elements by searching for regions of sequence homology shared between milk protein genes which respond similarly to lactogenic hormones and by searching for previously reported hormone receptor-binding sites. Within the conserved first 200 bp of 5' flanking sequences 3 regions of greater than 70% homology were observed between the rat beta- and gamma-casein genes. One of these contains a region 90% homologous to the chicken progesterone receptor-binding site. The conserved 5' noncoding region, the highly conserved signal peptide, and the hydrophobic carboxyl-terminal region of the protein are each encoded by a separate exon. In contrast the evolutionarily conserved phosphorylation site of beta-casein is formed by an RNA-splicing event. The exons which encode the phosphorylation sites of beta-casein appear to have resulted from an intragenic duplication. Based upon the exon structure of the casein genes, an evolutionary model of intragenic and intergenic exon duplications for this gene family is proposed.